Ruicheng Wei

Occurrence of veterinary antibiotics in animal wastewater and surface water around farms in Jiangsu Province, China

The objective of this investigation was to obtain a broad profile of veterinary antibiotics residues in animal wastewater and surface water around large-scale livestock and poultry farms in Jiangsu Province of China. Therefore, 53 samples collected from 27 large-scale animal farms in 11 cities and counties of Jiangsu Province in 2009, were monitored for 10 selected veterinary antibiotics using solid phase extraction and high performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) techniques. Ten veterinary antibiotics were found in animal wastewaters, eight antibiotics were detected in pond waters, and animal farm-effluents and river water samples were contaminated by nine antibiotics. The most frequently detected antibiotics were sulfamethazine (75%), oxytetracycline (64%), tetracycline (60%), sulfadiazine (55%) and sulfamethoxazole (51%) which were detected with a maximum concentration of 211, 72.9, 10.3, 17.0 and 63.6 μg L(-1), respectively. The maximum concentration of 0.55 μg L(-1) for cyromazine, 3.67 μg L(-1) for chlortetracycline, 0.63 μg L(-1) for sulfadoxine, 39.5 μg L(-1) for doxycycline and 0.64 μg L(-1) for sulfaquinoxaline were determined in the collected samples. In general, the maximum concentration of the selected veterinary antibiotics was detected in animal wastewaters except for chlortetracycline in animal farm-effluents. In addition, residue levels of selected veterinary antibiotics in animal wastewater and surface water around the farms were related to animal species and have a high spatial variation.

Occurrence of Ciprofloxacin, Enrofloxacin, and Florfenicol in Animal Wastewater and Water Resources

Antimicrobial agent residues are becoming an intractable environmental problem in soil, surface, and underground water. To obtain a broad profile of residues in animal wastewater and surface water, 24 animal wastewater, 8 animal farm effluent, 18 river water, and 8 pond water samples taken in Jiangsu in eastern China were monitored for enrofloxacin, ciprofloxacin, and florfenicol using solid phase extraction and high performance liquid chromatography/electrospray ionization-tandem mass spectrometry (HPLC/ESI-MS/MS) techniques. The results revealed that two antibacterials were detected simultaneously in 49.1% of samples, followed by three antibacterials (22.6%) and one antibacterial (22.6%). Up to 3.35, 5.93, and 2.10 μg L for ciprofloxacin, 1.09, 4.24, and 0.50 μg L for enrofloxacin, and 0.95, 2.40, and 2.84 μg L for florfenicol were detected in animal farm-effluent, river, and pond water, respectively. The maximum concentrations of ciprofloxacin and enrofloxacin in animal wastewaters were 7.49 and 8.77 μg L, respectively. Furthermore, residue levels of ciprofloxacin and florfenicol showed at least two statistical differences between any two sampling areas or two animal farms. Enrofloxacin showed no statistical difference among the sampling areas and the animal farms.

Occurrence of 13 veterinary drugs in animal manure-amended soils in Eastern China.

The occurrence of 13 veterinary drugs were studied in soil fertilized with animal manures in Eastern China. The 69 soil samples were obtained from twenty-three vegetable fields in 2009 and analysed for selected veterinary drugs by HPLC-MS/MS at soil depths of 0-20, 20-40 and 40-60 cm, and two additional samples were re-analysed from an earlier study from November 2011. Results showed that animal wastes, especially those from poultry farms, were one of pollution sources of veterinary drugs in soil. The detection frequency of veterinary drugs in soil was 83%, 91% and 87% in the three soil depths, respectively. The detection rates for the five classes of drugs in soils followed the rank order cyromazine > tetracyclines > sulfonamides > fluoroquinolones > florfenicol. Veterinary drugs were detected in soil layers at 20-40 and 40-60 cm depth to a greater extent than at 0-20 cm depth. The results of the same point in years 2009 and 2011 indicated that veterinary drugs accumulate easily and persist in the deeper soil. In addition, residue levels of veterinary drugs in soil were related to the animal species the manure was derived from. Overall, the predominance of tetracyclines in sampled soils underscored the need to regulate their veterinary use in order to improve the management and treatment of associated releases.

Characterization of NDM-5-positive extensively resistant Escherichia coli isolates from dairy cows

The aim of this study was to investigate the prevalence of blaNDM-5 gene in Escherichia coli isolates from dairy cows and to characterize the molecular traits of the blaNDM-5-positive isolates. A total of 169 cows were sampled (169 feces and 169 raw milk samples) in three dairy farms in Jiangsu Province and 203 E. coli isolates were recovered. Among these strains, three isolates carried blaNDM-5 gene, including one co-harboring mcr-1, which belonged to sequence type 446 and the other two belonged to ST2. Susceptibility testing revealed that the three blaNDM-5-positive isolates showed extensive resistance to antimicrobials. The blaNDM-5 gene was located on a ∼46-kb IncX3 transferrable pNDM-MGR194-like plasmid in all three isolates, while mcr-1 was located on a ∼260-kb IncHI2 plasmid pXGE1mcr. Competition experiments revealed that acquisition of blaNDM-5 or mcr-1-bearing plasmid can incur fitness cost of bacterial host, however, plasmid stability testing showed that both blaNDM-5 and mcr-1-carrying plasmid maintained stable in the hosts after ten passages without antimicrobial selection. Whole genome sequencing revealed that the mcr-1 gene coexisted with multiple resistance genes in pXGE1mcr and the backbone of this plasmid was similar to that of previously reported mcr-1-positive plasmid pHNSHP45-2. Moreover, pXGE1mcr could be conjugated into clinical NDM-5-positive E. coli isolates in vitro, thereby generating strains that approached pan-resistance. Active surveillance efforts are imperative to monitor the prevalence of blaNDM-5 and mcr-1 in carbapenem-resistant Enterobacteriaceae from dairy farms throughout China.

Molecular and virulence characterization of highly prevalent Streptococcus agalactiae circulated in bovine dairy herds

Bovine mastitis caused by Streptococcus agalactiae continues to be one of the major veterinary and economic issues in certain areas of the world. The more prevalent S. agalactiae strains that cause bovine mastitis in China dairy farms belong to a number of bovine-adapted sequence types (STs) ST67, ST103 and ST568. However, it is unknown why these STs can emerge as highly prevalent clones in bovine dairy farms. Here, to determine if a variety of virulence characteristics were associated with these highly prevalent STs, the molecular and virulence characterization of 116 strains isolated from bovine, human, fish and environment were analyzed. Our data showed that all bovine-adapted strains could be assigned to capsular genotype Ia or II, and carried pilus island 2b, and lactose operon. Importantly, we demonstrated that the growth ability in milk, biofilm formation ability and adhesion ability to bovine mammary epithelial cells (BMECs) were significantly higher for all bovine-adapted strains compared to strains from other origins. Additionally, ST103 and ST568 strains exhibited significantly higher hemolytic activity and cytotoxicity than ST67 strains. In conclusion, our study provides substantial evidence for the hypothesis that the virulence characteristics including efficient growth in milk, elevated biofilm formation ability, together with strong adhesion ability might have favored the high prevalence of the STs in the bovine environment, whereas the hemolytic activity and cytotoxicity were not the crucial characteristics.

A new, simple and rapid HPLC method for determination of chlortetracycline in pig solid manure

Chlortetracycline (CTC) is the most frequently used antibacterial in today’s swine production for disease treatment and growth promotion. It is not absorbed completely and a considerable amount is excreted into manure when CTC is administrated to animal. For this reason there is high concern on potential risks that manure-derived CTC enters into the surface water and selects for resistant microorganism. In order to support the monitoring of CTC and study its fate in manure and environment, a simple and rapid analytical procedure was developed to determine chlortetracycline (CTC) in pig solid manure. The extraction solution, composed of acetone-4mol.L−1 hydrochloric acid-water (13:1:6, v/v/v), was mixed into the manure sample, and the solution was pH adjusted at 2.0–2.2 before extraction for 20 min. Chromatographic separation was carried out on a C18 column at 375 nm with UV detector, using isocratic elution with oxalic acid-methanol-acetonitrile (72:14:14), v/v/v) as mobile phase at room temperature. Recoveries of CTC spiked samples at the levels of 1.0 mg.kg−1, 2.0 mg.kg−1 and 10.0 mg.kg−1 were 92.2%–100.73% with relative standard deviation (RSD) of 2.81–7.11% intra-day and 1.34–3.25% inter-day, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.2 mg.kg−1 and 0.8 mg.kg−1, respectively. The procedure developed in this study was tested to be simple, sensitive and suitable for the determination and screening of CTC in pig solid manure samples.

Study of intracellular Staphylococcus aureus control by virulent bacteriophage within MAC-T bovine mammary epithelial cells

ABSTRACT Bacteriophages (phages) are known to effectively kill extracellular multiplying bacteria. The present study demonstrated that phages penetrated bovine mammary epithelial cells and cleared intracellular Staphylococcus aureus in a time-dependent manner. In particular, phage vB_SauM_JS25 reached the nucleus within 3 h postincubation. The phages had an endocytotic efficiency of 12%. This ability to kill intracellular host bacteria suggests the utility of phage-based therapies and may protect patients from recurrent infection and treatment failure.

Occurrence of seventeen veterinary antibiotics and resistant bacterias in manure-fertilized vegetable farm soil in four provinces of China

This study focused on the occurrence of seventeen veterinary antibiotics and six resistant bacterias in soils from the vegetable farms fertilized with animal manure in China. Seventeen veterinary antibiotics, including sulfonamides, quinolones, tetracyclines, macrolides and amphenicols, were detected by high performance liquid chromatography/tandem mass spectrometer in all the 53 soil samples collected in four provinces during August 2016. The concentrations of target antibiotics in the soil samples ranged from not detectable to 415.00 μg/kg dry weight with the mean residual levels of the five classes followed order: tetracyclines (82.75 μg/kg) > quinolones (12.78 μg/kg) > macrolides (12.24 μg/kg) > sulfonamides (2.61 μg/kg) > amphenicols (0.06 μg/kg). Moreover, the highest antibiotic levels were found mainly in soil from organic vegetable farms. Risk assessment by using the methods of risk quotient, suggested that oxytetracycline, chlortetracycline, enrofloxacin and ciprofloxacin could pose severe ecological risk in sampled soils. Resistant strains were isolated in 30 samples, with Escherichia coli and Klebsiella pneumonia found the dominant bacterial hosts with resistance genes. Antibiotic resistance genes, including tetA, tetB, qnrS, oqxA, sul1, sul2, ermA and floR, were detected in the strains resistant to: tetracyclines, quinolones, sulfonamides, macrolides and amphenicols resistance, respectively. Overall, there was a correlation between the results of antibiotic risk assessment with the detection of resistance genes from isolated strains in the soils.

Corrigendum: Staphylococcus aureus bacteriophage suppresses LPS-induced inflammation in MAC-T bovine mammary epithelial cells

Several previous studies have shown that bacteriophages can significantly affect the production of various cytokines. The aim of this present study was to investigate the inflammatory effects and mechanisms of bacteriophage vB_SauM_JS25 in stimulated MAC-T bovine mammary epithelial cells by real-time polymerase chain reaction (PCR) and Western blotting. Experiments show that vB_SauM_JS25 reduces Staphylococcus aureus- or lipopolysaccharide (LPS)-induced levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-8, IL-10, and regulated on activation, normal T cell expressed and secreted (RANTES) mRNA in MAC-T cells, in a manner expected to be unrelated to its antibacterial action. Moreover, S. aureus bacteriophage vB_SauM_JS25 suppressed the LPS-induced phosphorylation of nuclear factor (NF)-κB p65, which may represent an important mechanism mediating these effects. A carefully regulated balance between activation and inhibition by bacteriophages must be kept avoiding inappropriate inflammatory responses. The ability of vB_SauM_JS25 to influence the immune response highlights the potential development and application of bacteriophage-based therapies and may represent a novel anti-inflammatory therapeutic strategy.

Insights Into the Bovine Milk Microbiota in Dairy Farms With Different Incidence Rates of Subclinical Mastitis

Bovine mastitis continues to be a complex disease associated with significant economic loss in dairy industries worldwide. The incidence rate of subclinical mastitis (IRSCM) can show substantial variation among different farms; however, the milk microbiota, which have a direct influence on bovine mammary gland health, have never been associated with the IRSCM. Here, we aimed to use high-throughput DNA sequencing to describe the milk microbiota from two dairy farms with different IRSCMs and to identify the predominant mastitis pathogens along with commensal or potential beneficial bacteria. Our study showed that Klebsiella, Escherichia–Shigella, and Streptococcus were the mastitis-causing pathogens in farm A (with a lower IRSCM), while Streptococcus and Corynebacterium were the mastitis-causing pathogens in farm B (with a higher IRSCM). The relative abundance of all pathogens in farm B (22.12%) was higher than that in farm A (9.82%). However, the genus Bacillus was more prevalent in farm A. These results may be helpful for explaining the lower IRSCM in farm A. Additionally, the gut-associated genera Prevotella, Ruminococcus, Bacteroides, Rikenella, and Alistipes were prevalent in all milk samples, suggesting gut bacteria can be one of the predominant microbial contamination in milk. Moreover, Listeria monocytogenes (a foodborne pathogen) was found to be prevalent in farm A, even though it had a lower IRSCM. Overall, our study showed complex diversity between the milk microbiota in dairy farms with different IRSCMs. This suggests that variation in IRSCMs may not only be determined by the heterogeneity and prevalence of mastitis-causing pathogens but also be associated with potential beneficial bacteria. In the future, milk microbiota should be considered in bovine mammary gland health management. This would be helpful for both the establishment of a targeted mastitis control system and the control of the safety and quality of dairy products.